SARS-CoV-2混合稀释样本核酸检测效能分析

Analysis of nucleic acid detection efficiency of SARS-CoV-2 mixed and diluted samples

目的评价混合稀释样本在新型冠状病毒(severe acute respiratory syndrome Coronavirus 2,SARS-CoV-2)核酸检测的效能,为大规模人群的SARS-CoV-2核酸检测提供参考方案。方法于2021年3月至2021年11月分别采用有固定Ct值靶值的国家卫健委SARS-CoV-2阳性质控和第三方试剂公司的SARS-CoV-2弱阳性质控样本,用阴性样本分别以1∶5、1∶10、1∶15、1∶20进行稀释。以实时荧光定量聚合酶链反应(real-time fluorescence quantitative PCR,RT-qPCR)检测原始及各稀释度样本SARS-CoV-2双靶标ORF1ab基因和N基因,通过统计学分析混合样本检测效能。结果阳性与弱阳性质控样本ORF1ab基因和N基因检测Ct值随样本稀释倍数增大而不断增大,各组检测Ct值均值一致性差异有统计学意义,阳性与弱阳性质控样本ORF1ab和N基因P值分别为0.015、0.013、0.021、0.023,均P<0.05。阳性与弱阳性质控样本ORF1ab基因和N基因阳性检出率随样本稀释倍数增加而下降。阳性质控样本在1∶20稀释时出现阴性检测结果;弱阳性质控样本超出1∶10稀释时出现阴性检测结果,且随着稀释倍数的增大超出试剂检测限,出现Ct值检测不出现象。阳性与弱阳性质控样本两靶标基因检测Ct值与稀释倍数之间相关系数均在0.9以上,呈高度正相关。结论进行大范围人群SARS-CoV-2核酸检测筛查时,混合稀释样本应尽可能选择低倍稀释,不应超过10倍稀释,低于试剂检测限可造成假阴性结果。

Objective To evaluate the efficacy of mixed dilution samples in the detection of SARS-CoV-2 nucleic acid and provide a reference framework for large-scale population testing.MethodssFrom March 2021 to November 2021,the SARS-CoV-2 positive quality control samples from the National Health Commission and weak positive quality control samples from a third-party reagent company with fixed Ct target values were used,and negative samples were diluted at ratios of 1:5,1:10,1:15,and 1:20,respectively.Real-time fluorescence quantitative PCR was used to detect the original samples and the dual target genes,open reading frame lab(ORFlab)gene and nucleocapsid protein(N),gene of SARS-CoV-2 in each dilution sample,and the detection efficiency of the mixed samples was analyzed statistically.Results The Ct values of the ORFlab gene and the N gene of positive and weak positive quality control samples increased with the increase of sample dilution times,and the mean consistency difference of Ct values in each group was statistically significant.The P values of the ORFlab gene and the N gene in positive and weak positive quality control samples were 0.015,0.013,0.021,and 0.023,respectively,with all P<0.05.The positive detection rate of the ORFlab gene and the N gene in positive and weak positive quality control samples decreased with the increase of sample dilution.The positive quality control sample showed negative test results at 1:20 dilution.Weak positive quality control samples showed negative test results beyond 1:10 dilution,and with the increase of dilution beyond the detection limit of the reagent,the Ct value could not be detected.The correlation coefficients between the Ct value and dilution times of the two target genes detection in positive and weak positive quality control samples were both above O.9,showing a highly positive correlation.Conclusion When screening a large-scale population of SARS-CoV-2 nucleic acid detection,the mixed dilution samples should be selected as low as possible,and should not exceed 10 times dilution,which may cause false negative results below the detection limit of reagents.